The present invention relates to the fields of molecular biology and microbiology and the use of genetic techniques to modify existing enzymatic pathways for the production of desired compounds. More specifically, the present invention relates to a method for the production of para-hydroxybenzoate (PHBA) by a toluene-metabolizing Pseudomonas mendocina mutant lacking the ability to express para-hydroxybenzoate hydroxylase (PHBH).
Para-hydroxybenzoate (PHBA) is the key monomer for Liquid Crystal Polymers (LCPs) which contain approximately 67% PHBA. Esters of PHBA also can be used as backbone modifiers in other condensation polymers, i.e., polyesters, and are also used to make parabens preservatives.
Chemical synthesis of PHBA is known. For example, JP 05009154 teaches a chemical route using the Kolbe-Schmidt process from tar acid and CO2 involving the extraction of tar acid from a tar naphthalene oil by an aqueous potassium hydroxide; adding phenol to the extracted tar acid potassium salt; removing H2O and reacting the resultant slurry with CO2. Alternative methods of synthesis are also known; see, for example, U.S. Pat. No. 5,399,178; U.S. Pat. No. 4,740,614; and U.S. Pat. No. 3,985,797.
Chemical synthesis is problematic and costly due to the high energy needed for synthesis and extensive purification of product required. An alternate low cost method with simplified purification would represent an advance in the art. Biological production offers one such low cost, simplified solution to this problem.
Microbiological methods of PHBA synthesis are known. For example, JP 06078780 teaches PHBA preparation by culturing benzoic acid in the presence of microorganisms (preferably Aspergillus) that oxidize benzoic acid to PHBA.
An alternate method of biological production is suggested by bacteria that have an enzymatic pathway for the degradation of toluene and other organics where PHBA is produced as an intermediate. The first enzyme in the toluene degradation pathway is toluene monooxygenase (TMO) and the pathway is referred to as the TMO pathway. Bacteria that possess the TMO pathway are primarily restricted to the genus Pseudomonas where P. putida, P. fluorescens, P. aeruginosa and P. mendocina are the most commonly utilized species. The TMO pathway has been highly characterized [Romine et al., Bioremediation of Chlorinated Polycyclic Aromatic Hydrocarbon Compounds (1994), 271-6. Editor(s): Hinchee, Robert E. Publisher: Lewis, Boca Raton, Fla.] and a number of the genes encoding key enzymes have been cloned and sequenced, including the protocatechuate 3,4-dioxygenase genes [Frazee, J. Bacteriol., (1993), 175(19), 6194-202], the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate [Romero-Steiner et al., J. Bacteriol. (1994), 176(18), 5771-9; Dimarco et al., J. Bacteriol. (1994), 176(14), 4277-84] and the pobA gene encoding the expression of para-hydroxybenzoate hydroxylase (PHBH), the principal enzyme for the conversion of PHBA to protocatechuate [Wong et al., Microbiology (Reading U.K.) (1994), 140(10), 2775-86; Entsch et al., Gene (1988), 71(2), 279-91].
Bacteria that possess the TMO pathway are useful for the degradation of toluene and trichloroethylene and are able to use these and other organics as sole carbon sources where they are transformed through PHBA to ring opening degradation products (U.S. Pat. No. 5,017,495; U.S. Pat. No. 5,079,166; U.S. Pat. No. 4,910,143).
Recently, various strains of Pseudomonas possessing the TMO pathway have been used to produce muconic acid via manipulation of growth conditions (U.S. Pat. No. 4,657,863; U.S. Pat. No. 4,968,612). Additionally, strains of Enterobacter with the ability to convert p-cresol to PHBA have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce PHBA from p-cresol.
Although the above cited methods are useful for the production of PHBA, these methods are limited by the high cost and toxicity of the aromatic substrate, p-cresol. Furthermore, the above methods use an isolated wildtype organism that converts part of the p-cresol to PHBA while the rest is further metabolized. The utility of these methods are therefore limited by low yields and an inability to control further degradation of the desired product.
Shuman et al., (J. Biol. Chem., (1993), 268, 17057) have reported the identification of a gene encoding a putative isozyme of PHBH from P. fluorescens. The comparison of the deduced amino acid sequence from this gene with the enzyme encoded by the more fully characterized pobA gene of P. fluorescens (van Berkel et al., Eur. J. Biochem., (1992), 210 (2), 411-419) demonstrates 73% homology with the isozyme. Although the work of Shuman et al. (supra) suggest the presence of a second gene encoding an alternate PHBH, the authors were not able to isolate any expressed protein and concluded that the gene encoding the isozyme is not expressed.
Therefore, the problem to be overcome is to develop a method of microbially mediated PHBA production from an inexpensive, aromatic substrate, where the microbial TMO pathway has been altered to prevent the degradation of PHBA.
The present invention provides a method for the production of PHBA comprising: (i) culturing a pobA(xe2x88x92) Pseudomonas strain in a medium containing an aromatic organic substrate, at least one suitable carbon source, and a nitrogen source, wherein the pobA(xe2x88x92) Pseudomonas strain comprises genes encoding the TMO toluene degradation pathway and wherein the pobA(xe2x88x92) Pseudomonas strain does not produce any detectable para-hydroxybenzoate hydroxylase, whereby PHBA accumulates; and (ii) recovering the PHBA.
The invention further provides genes encoding novel para-hydroxybenzoate hydroxylase enzymes and pobA(xe2x88x92) Pseudomonas strains deficient in hydroxybenzoate hydroxylase that are able to accumulate PHBA under suitable growth conditions and in the presence of a suitable aromatic substrate.